Technical Abstract:
Control of insects that vector pathogens is a massive challenge to human health and agriculture. Yellow dwarf viruses (YDV) cause economically significant disease in cereal crops (barley, wheat, rye, maize) worldwide and are vectored by aphids. The identification of vector proteins mediating virus transmission is critical to develop agriculturally sustainable virus management practices and to understand arbovirus (arthropod-borne virus) strategies for movement in all insect vectors. Previously, we used a top-down approach (DIGE) to describe heritable proteome variation in aphid vector (competent to transmit viruses) and non-vector (does not transmit viruses) genotypes. Vector-specific isoforms were predictive of the transmission phenotype. We sought to identify the sources of heritable proteome variation and to understand how these proteins function in virus transmission.
METHODS
Transcripts encoding for protein isoforms identified by DIGE-LC-MS/MS were cloned, sequenced, and aligned using BLAST to identify polymorphisms between vector and non-vector aphids. Isoform-specific expression was verified in aphids using selected reaction monitoring targeting polymorphic peptides. To test for functional interactions, YDV-aphid protein complexes were co-immunoprecipitated from aphid homogenate using anti-YDV antibodies and magnetic beads. Protein complexes were eluted, reduced, alkylated, and digested with trypsin. Nanoflow LC-MS/MS was performed using a Q-Exactive. Peptide assignments were made using an aphid RNAseq database and quantified using MS1 peak area integration in Skyline. Control co-immunoprecipitation reactions were thoroughly characterized using the same LC-MS/MS method to rule out non-specific contaminants. Direct interactions with YDV are being validated in vitro using his-tagged fusion proteins.
PRELIMINARY DATA
cDNA sequencing revealed cis-acting mutations in genes with multiple isoforms. Mutations conferred single amino acid polymorphisms that conferred a change in charge of the protein, an observation supported by previous isoelectric focusing data. Selected reaction monitoring verified the expression of these protein isoforms in vector and non-vector aphids, indicating that a targeted proteomics approach could be developed to rapidly screen insect populations and predict vectoring capacity on the basis of expression of multiple, vector-specific isoforms. Co-immunoprecipitation (CoIP) revealed that a number of these proteins, as well as previously uncharacterized proteins interact directly or in complex with purified YDV. Two classes of proteins were identified: those specific to the coIP and those enriched as compared to control co-IP reactions with no virus. CYCLOPHILIN, a prolyl isomerase, which catalyzes cis-trans isomerization in proline residues, was identified as a YDV-interacting partner in the co-IP in multiple aphid genotypes. Very few spectra from CYCLOPHILIN were detected in control co-IP reactions, and these may be attributed to carry over between LC-MS/MS runs due to the randomization incorporated into the study design. Importantly, all YDV species and related viruses contain a proline-rich domain known as the proline hinge that is crucial to providing flexibility and accessibility of a minor structural protein involved in virus-plant and virus-aphid interactions. cDNA sequencing analysis revealed a single amino acid polymorphism in CYCLOPHILIN in more than twenty aphid genotypes linked to vectoring capacity. Parallel coIP studies in our lab to identify virus-interacting plant proteins revealed the plant homolog of CYCLOPHILIN as an interacting partner of a closely related virus (Potato leafroll virus), indicating CYCLOPHILIN may be universally involved in protein folding and transport of YDV and related viruses. Direct interaction of aphid CYCLOPHILIN to purified YDV was confirmed in vitro.
NOVEL ASPECT
Mass spectrometry coupled to molecular biology revealed an evolutionarily conserved arbovirus movement strategy in both host and insect vector.